EFFECTS OF ENVIRONMENTAL FACTORS ON NUTRIENTS AND ANTINUTRIENT CONTENTS OF SELECTED LEAFY VEGETABLES A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN AGRONOMY AND SOIL SCIENCE MAY 1986 By Patma Vityakon Dissertation Comnittee: Goro Uehara, Chairman Robert L. Fox James A. Silva Russell S. Yost Robert E. Pauli Bluebell R. Standal We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Agronomy and Soil Science. DISSERTATION COMMITTEE Chairman ^ c u c l £ . Dedicated to my Father ACKNOWLEDGMENTS My thanks go to the members of my dissertation committee, especially to Dr. Goro Uehara and Dr. Bluebell Standal; the staff of the Benchmark Soils Project; the staff of the Department of Agronomy and Soil Science, University of Hawaii; the staff of the East-West Center, especially those at the Environment and Policy Institute; my colleagues and friends in Hawaii and far away; and my family in Thailand, especially my mother. With their unfailing interest, assistance and encouragement, I have completed this work in good spirits. The sponsorship of the East-West Center and the partial funding of my studies in Hawaii received from the Government of Thailand are gratefully acknowledged. This dissertation was skillfully typed by Freda Hellinger. ABSTRACT The purpose of this research was to test the hypothesis that plant composition in general, and plant antinutrient content in particular, are affected by environmental factors. To test this hypothesis three crops were grown in four benchmark locations which had been character­ ized for soils and provided with weather stations to monitor air and soil temperatures, relative humidity, rainfall, solar radiation and wind speed. The four experimental sites represented four soil series and two soil families. The Wahiawa and Lahaina soil series identified on sites on the Islands of Oahu and Molokai were members of the clayey, kaolinitic, isohyperthermic family of Tropeptic Eutrustox, whereas, the Niulii and Kukaiau soil series identified on sites in the Kohala and Hamakua districts of the Big Island of Hawaii were members of the thixotropic, isothermic family of Hydric Dystrandepts. Three test crops were used to test the hypothesis: amaranth (Amaranthus gangeticus L.), a crop cultivated for its tender leaves or grain, cassava (Manihot esculenta L.), a crop normally cultivated for its starchy tubers, and taro (Colocasia esculenta L. (Schott.), a crop normally grown for its underground corms. The leaves of all three crops are consumed by people in the warm tropics. For this reason, the leaves of all three crops were sampled and analyzed to measure the effects of soil and climate variables on oxalate, nitrate , and ionic contents of leaves. Amaranth experiments were installed at three sites. At each site, irrigated and non-irrigated experiments were conducted. Within each irrigation experiment, three fertilizer treatments consisting of (1) a basal treatment of lime, N, P, K, bases, and trace nutrients, (2) a N treatment superimposed on the basal treatment, and (3) a P treatment superimposed on the basal treatment, were arranged in a randomized com­ plete block design with three replications. Plant tops were harvested at maturity for chemical analyses. Cassava leaves were sampled from ongoing experiments at the four experimental sites. Taro leaves were also sampled from ongoing experiments but from only three sites. Soil and climatic factors significantly influenced the chemical compositions of crops. These effects differed for each crop. Nitrogen plays an important role in controlling the synthesis of oxalate and the accumulation of nitrate in amaranth. The highly variable oxalate and nitrate contents in plants grown in environmentally different sites were, to a large extent, due to different soil N contents. Virtually all oxalate in cassava was in the form of calcium oxalate, so that tissue Ca content was an important factor in oxalate formation. In taro, K appeared to be the key factor accounting for the difference in oxalate content among sites. It was concluded from the results of this study that plant com­ positions can be controlled by management of the environment and crop selection. It follows from this conclusion that the nutritional quality of food crops can be measurably improved if more research is directed towards achieving this goal. TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................... m ABSTRACT ................................................. iv LIST OF TABLES ........................................... X LIST OF FIGURES ........................................... xvii LIST OF ABBREVIATIONS .................................... XX i CHAPTER I INTRODUCTION .............................. 1 CHAPTER II REVIEW OF LITERATURE ..................... 2.1 Anti nutrient oxalate and toxic agent nitrate in foods ..................... 2.2 Oxalate accumulation in vegetables and other plants and its effects on the health of humans and animals ......... 2.3 Factors affecting oxalate contents in plants ............................ 12 2.3.1 The concept of cation-anion balance ........................ 12 2.3.2 Soil ionic environments .... 13 2.3.3 Environmental factors ........ 16 2.3.4 State of growth or maturity . 16 2.3.5 Plant genetics ............... 17 2.4 Nitrate accumulation in vegetables and its effects on human and animal health 18 2.5 Factors affecting nitrate accumulation in vegetables ........................ 22 2.5.1 Nutrient supply ............... 23 2.5.2 Genetic factor ............... 24 2.5.3 Environmental factors ........ 25 2.6 Relationship between total N and nitrate-N concentration in plants . 27 2.7 Chemistry of oxalate with an emphasis on its biosynthesis in plants . 28 v n 2.8 Relation between oxalate synthesis, nitrate accumulation and nitrogen metabolism in plants ....................... 32 2.9 The use of amaranth, cassava and taro as vegetables ............................. 37 2.9.1 Distributions of crop and their u s e s ............................... 37 2.9.2 Nutrition values and some limitations......................... 39 2.10 Estimation of oxalate contents ............. 45 CHAPTER III MATERIALS AND METHODS ........................... 47 3.1 The experimental s i t e .................... 47 3.2 C r o p s ..................................... 50 3.3 Experiments .............................. 57 3.3.1 Amaranth field experiments ........ 57 3.3.2 Cassava............................ 63 3.3.3 T a r o .............................. 64 3.4 Laboratory Analyses ...................... 66 3.4.1 P l a n t ............................... 67 3.4.2 S o i l s ............................... 69 3.5 Data a n a l y s i s ......................... 70 3.6 Development of methods for oxalate extraction, fractionation and determination ............................. 72 3.6.1 Extraction of total oxalate from plant t i s s u e ....................... 72 3.6.2 Extraction of oxalate fractions . 73 3.6.3 Preparation of the samples from the different oxalate fraction for HPLC analysis....................... 74 3.6.4 Description of oxalate determination by HPLC technique................... 75 3.6.5 Comparison of the classical and HPLC methods of oxalate determination............ 77 3.6.6 Oxalate recovery from the extraction and fractionation of plant materials........................... 84 v m 3.7 Soil and weather characteristics measured specifically for the study of agro- environmental effects on nutritional quality of food c r o p s ................ 89 CHAPTER IV RESULTS AND DISCUSSION ......................... 97 4.1 Growth and development of amaranth... 98 4.1.1 Energy utilized by amaranth for emergence and g r o w t h .............. 98 4.1.2 Dry w e i g h t ........................ 100 4.1.3 Moisture content of plant tissue . 105 4.2 Oxalate compositions of amaranth tissue . 107 4.2.1 Forms and amount of oxalate in amaranth......................... 107 4.2.2 Oxalate and calcium concentrations in amaranth t i s s u e ................ 116 4.2.3 Comparison of chemical composition in plants grown at different s i t e s ............................... 116 4.2.4 Effect of irrigation on plant chemical composition .............. 133 4.2.5 Effects of N and P fertilizers on plant chemical composition ........ 139 4.2.6 Relationships between oxalate, nitrate and agroenvironmental factors........................ 155 4.3 Response of cassava to environmental f a c t o r s .............................. 186 4.3.1 Forms and content of oxalate in cassava leaves ..................... 193 4.3.2 Chemical compositions of plants grown in different s i t e s ..... 198 4.3.3 Effects of irrigation on chemical compositions of plants grown in Molokai and Waipio sites .......... 203 4.3.4 Influence of plant, soil, and climatic factors on oxalate and nitrate concentrations ..................... 204 4.4 Response of taro to environmental factors. 209 4.4.1 Forms of oxalate in t a r o ..... 209 IX 4.4.2 Comparison of chemical composition of plants grown in different sites ............................. 215 4.4.3 Relationships between oxalates in taro leaves and soil and climatic variables ......................... 223 CHAPTER V SUMMARY AND CONCLUSIONS ....................... 226 APPENDICES ................................................. 231 A. Analysis of variance of various